There are an estimated 1.5 million cases of acute coronary events in the U.S. annually. Most are caused by occlusion of coronary arteries and 40% are fatal. Treatment usually involves mechanical or enzymatic removal of the occlusion, and reperfusion of the ischemic heart. It has been recognized for some time that reperfusion promotes additional cardiac injury and cell death that exacerbates the process of infarction. As a consequence, there is great interest in identifying the molecular basis of the damage and developing methods to prevent or reduce infarction. Two approaches are under intense scrutiny as a means to treat myocardial infarction, one involves cardioprotection to reduce injury at the time of reperfusion, and the other involves attempts to repair the damage by introducing stem cells. The current application adheres to the thesis that both of these approaches are of equivalent importance. Studies in multiple species including rodents, rabbits, dogs, and pigs have demonstrated that the size of an infarct caused by ischemia/reperfusion is reduced 50% or more when apoptosis is inhibited. Multiple cardioprotective strategies converge at the level of the BH3-only pro-apoptotic proteins, where the irreversible death/survival decision is made. These proteins represent the final frontier for anti-apoptosis therapy and offer targets of high specificity. Bnip3 has emerged as a central regulator of cell death during both ischemia and reperfusion, yet we know virtually nothing about its mechanism of action, how it is activated, or how it may be most effectively inhibited. We propose here to test 2 main hypotheses: firstly, Bnip3 is a phosphoprotein that requires a PKC-?-dependent step for activation; secondly, Bnip3 activates the mitochondrial permeability transition pore independently of other BH3-only proteins by interacting with cyclophillin-D. The application has 5 aims: (1 & 2) To define the roles of phosphorylation in the molecular regulation of Bnip3 and identify the kinase(s). (3) To determine the pathway of Bnip3-mediated death under conditions of ischemia alone and ischemia with reperfusion. (4) Define the relationships and functional hierarchy of Bnip3 within other BHS-only proteins (Bid, Bad, PUMA, NOXA) that are implicated in promoting infarction. (5) Define a functional region of the Bnip3 N-terminus that is cardioprotective when the transmembrane domain is deleted. Successful achievement of these aims will provide a more complete characterization of the role of Bnip3 in myocardial infarction and test a new molecular approach to therapy for reperfusion injury. [unreadable] [unreadable] [unreadable]